Tuesday, July 27, 2010

I was told by Sarah that if I did not write on the blog of the esteemed MacQueen lab, we could not be best friends. Therefore, I must post!

A few members of the MacQueen lab tentatively came into the Hingorani lab the other day to look at my microplates. In the microplates were fabulous colors (whose beauty was magnified by the photography skills of the MacQueen gang--see 'Sourjourn into Manjuland").

I am working on characterizing the kinetics of three MutS (Msh2-Msh6 in humans) mutants. MutS is a protein that recognizes mismatched DNA and recruits other proteins to initiate the DNA mismatch repair process. I am studying three cancer-associated mutants in Thermus aquaticus ("Taq"), as it is an easy organism to work with and there is high sequence homology between Taq and human MutS. In addition to binding mismatched DNA, MutS it is also an ATPase, and we're interested in the mechanism of ATP hydrolysis (ATP--> ADP + Pi) in the MutS mutants, and it is thought that MutS may communicate that it has bound a mismatch through coordination of its DNA binding site and ATPase domain.

One way to measure ATP hydrolysis is through phosphate production. The microplates that the MacQueeners photographed were part of the "Malachite Green Assay." Malachite green turns from yellow to green when it binds phosphate and this color change can be quantified. We create a standard curve with our stock of phosphate and then use this standard to calculate the concentration of phosphate at different time points in the reaction mixture that includes MutS and ATP. Using the concentration of phosphate at different time points in the reaction, we are able to determine kcat, which informs us about how fast the protein hydrolyzes ATP (more formally, kcat represents how many molecules of ATP are hydrolyzed per MutS active site per minute).

3 comments:

That was so informative! thanks so much for writing that entry. This was very exciting because I worked with mutS bacteria (unsuccesfully) last semester so I was very excited to see an unexpected word that I recognized. Also when I saw the titile I though it was an entry by Sarah bc it said "bffls!" OMG!